1. Field of the Invention
The present subject matter relates to stem cell collection from the placenta detailing a new method which is clinically feasible, convenient, and highly efficient. It also involves making harvesting and banking of all tissues derived from the placenta easy, efficient, under the highest quality control by delivering the clamped unmanipulated cooled sterile packaged placenta post-partum to a potential centralized facility that can service a local, state, country, region, or world-wide customer/patient base. Particularly, the present subject matter describes a new finding that residual placenta cord blood cells gathered by machine pulsatile perfusion are more enriched with the primitive hematopoietic stem cell phenotypes (CD133+) compared to those from conventional needle/syringe withdrawal or gravity drainage collection and potentially allows cord blood cells from a single donor to be used for regenerative medicine purposes through out a lifetime. Increased stem cell numbers obtained from a single placenta using the described method can also improve allogeneic hematopoietic stem cell transplant outcomes and obviate the use of double or triple cord blood grafts from different donors in order to compensate for insufficient stem cells from a single donor graft. Another particular of the present subject matter is the new finding that a whole unmanipulated placenta right after delivery with the umbilical cord clamped can be placed into a suitable sterile container that is cooled with ice and transported to a central facility up to 40 hours away for harvesting and banking of all available tissue types without any significant loss of viability.
2. Description of the Background Art
Cord Blood Collection Method and Cord Blood Transplantation in Adults
Umbilical CB cells are a promising source of HSC to perform allogeneic HSC transplantation for hematological malignancies and bone marrow failure syndrome (Kurtzberg et al., 1996; Wagner et al., 1996; Gluckman et al., 1997; Rubinstein et al., 1998). Significant advantages include a rapid access to CB cells which are stored in CB banks nationwide and acceptance of 1-2 human leukocyte antigen mismatch grafts due to infrequent severe graft versus host disease (GVHD) compared to the matched unrelated donor grafts (Barker et al., 2002). CB cells enable patients to choose allogeneic transplant as a curative option for hematological malignancies where otherwise no suitable match donors are available, particularly among patients in minority groups. Despite the above advantages, the use of CB is limited in adults due to insufficient numbers of cells, including CD34+ cells and progenitors. CB transplant using low levels of total nucleated cell counts leads to significant delays in post-transplant engraftment of neutrophils and platelets or engraftment failures (Wagner et al., 2002; Laughlin et al., 2004). Known procedures for harvesting CB include draining the blood by gravity from the delivered placenta, and draining the blood by venipuncture into collection bags or syringes.
Since CB supplies are barely enough for only one time use or more recently using double CB supplies from two non-identical donors, adult CB transplants have been performed generally under a clinical research basis only when suitable unrelated donors are not available. In practice, a recovery of only 20-40 ml is not unusual and these CB cells are therefore not even used or stored (Lasky et al, 2002; George et al, 2006). In such cases, a significant amount of uncollected CB cells still remain in the placenta and are discarded since there is no standardized supplemental method that can collect them after the initial harvest to supplement it. To expand the future CB bank donor pool, it is important to investigate improved CB harvesting methods including how to collect the residual CB cells that are left after conventional CB harvesting (Harris et al, 1994). More importantly, availability of an increased amount of CB cells from the same placenta may allow storing an amount of CB cells sufficient for multiple uses including back up or graft engineering such as an ex vivo expansion and adoptive immunotherapy.
Current Knowledge in HSC Plasticity and Tissue Regeneration
Over the past decade, many types of stem cells which have the capacity to replicate, self-renew, and differentiate, have been identified in humans. Totipotent stem cells are capable of forming every type of body cell, and these cells are within the early embryo and are the so-called human ES cells. Pluripotent stem cells are capable of developing into endoderm, mesoderm, or ectoderm. Tissue specific stem cells are committed to make certain tissues only. For example, hematopoietic stem cells (HSC) are responsible for all types of blood cells but no other tissue types and their continued presence in an adult allows for a repair capability. However, investigators have found that cells like adult HSC which were considered to be responsible for production of different types of hematopoietic progenitor cells even gave rise to cells of different tissue or organ such as neural cells or muscle cells.
Research studies on transdifferentiation of adult HSC continue to be controversial and active research investigations are on going. In contrast, a number of clinical cases have reported evidence of nonhematopoietic cell generation after either BM transplantation or cardiac transplantation. A retrospective study to look for BM transdifferentiation into brain after BM transplantation showed evidence of neuropoiesis, detection of astrocytes and microglia in a long-term setting without cell fusion (Cogle et al., 2004). Other reports have noted detection of donor cells in osteoblasts, hepatocytes, gastro-intestinal (GI) tract epithelia, stroma after BM transplant; keratinocytes/hepatocytes/GI tract/skin epithelia after peripheral blood stem cell transplant; and cardiomyocytes with and without endothelium after cardiac transplant with a wide range of percent amounts found (Hruban et al., 1993; Theise et al., 2000; Korbling et al., 2002; Muller et al., 2002; Okamoto et al., 2002; Quaini et al., 2002).
Cord Blood Cells as a Source of Adult Stem Cells
Although human ES cells can be differentiated and expanded in vitro to produce different types of progenitors, its application in patients is currently hindered by multiple ethical issues. In addition, the purity issue of embryonic stem cell-derived progenitor cells has to be solved. By contrast, adult stem cell populations derived from hematopoietic tissues including bone marrow and umbilical CB cells were found to be capable of differentiation into ectoderm or endoderm upon exposure to adequate stimuli (Eglitis and Mezey, 1997; Brazelton et al., 2000; Mezey et al., 2000; Sanchez-Ramos et al., 2001; Chen et al., 2005). In particular, CB derived stem cells have further advantages compared to the other sources since they are collected from the placenta which is normally discarded, thus requiring no tissue damage to the host upon harvesting the cells. Compared to the BM cells, CB has primitive ontogeny with naïve immune status and relatively unshortened telomere length.
Among debates concerning whether truly pluripotent somatic stem cells exist, cells derived from the CB and placenta have been increasingly focused on as containing interesting properties for potential clinical exploitation. Recently, CB has been shown to contain a heterogeneous cell population and recognized as a source of pluripotent stem cells (Goodwin et al, 2001; Sanchez-Ramos et al, 2001; Bicknese et al, 2002; Sanchez-Ramos, 2002; Zigova et al, 2002) Others reported that adherent cell population isolated from a week long suspension culture of CB cells after lineage positive cell depletion were shown to express immunohistochemical evidence of ectodermal and endodermal features (McGuckin et al, 2004). There is a series of successful CB transplant reports on children suffering from the neurodegenerative disorder Krabbe leukodystrophy (Escolar et al, 2005).
The present inventor hypothesizes that these unique features found in CB stem cells may be from a recently published emerging concept that the gestational placenta may be a hematopoietic niche during embryo development (Gekas et al, 2005; Ottersbach and Dzierzak, 2005). It is a simple assumption that a full-term placenta may also contain remnant primitive stem cells adherent to the vascular niche, or possibly due to the stress associated with “birth”, there may be an increased number of circulating stem cells that are released from fetal BM or liver which have migrated to the placenta niche. Thus, the present inventor hypothesizes that placenta derived CB cells obtained by this innovation may contain more primitive stem cells (ES cell-like cells) left as remnant stem cells deposited in a placenta vascular bed niche since embryogenesis.
Isolation and Selection of Primitive CB Cells Including ES Cell-Like Cells and Primitive HSC
To identify common stem cell markers using comparison analysis of gene expression patterns from embryonic, hematopoietic, and neural stem cells, only one gene was identified (probably due to technical difficulties) (Fortunel et al, 2003). Thus, to identify and select stem cells may still require several markers to isolate these cells. One of the characteristics that can be used to distinguish stem cells is the absence of markers of differentiation. This approach has been used widely in HSC field to perform enrichment of stem cells to be employed for therapy. This “Lineage negative (Lin−)” trait is a common property of many stem cell populations (Cai et al, 2004b). To further enrich the stem cell population from Lin− CB cells, it has been reported that CD133+ marker demonstrated a high proliferation potential on growth factor stimulation (Forraz et al, 2004). Others reported that CD133+/CD34− subset might represent more primitive stem cells as they did not produce colony forming cells (CFC) in methylcellulose, but exhibited the highest SCID repopulating cells frequency (Kuci et al, 2003). Embryonic stem cell markers such as stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81 are expressed only on ES cells which have been widely used in the characterization of pluripotent stem cell and antibodies compatible to FACS analysis (which are commercially available). Most recently, Kucia et al. described a primitive stem cell population called “very small embryonic-like (VSEL) stem cells” which carry Lin−/CD45−/CXCR4+/CD133+/CD34+ phenotype (Kucia et al, 2006). These cells were also positive for embryonic transcription factors Oct-4 and Nanog.
Alternatively, the method using the presence of general metabolic markers has also been used to identify and isolate stem cells. One of the metabolic markers that has been described is aldehyde dehydrogenase (ALDH) (Takebe et al, 2001). The fluorescent substrate of ALDH, Aldefluor (StemCell), has been used to demonstrate increased ALDH activity in neural stem cells (Cai et al, 2004b; Corti et al, 2006) and HSC (Storms et al, 1999). This nontoxic, live labeling method can be used to identify other stem cell populations as well (Cai et al, 2004a). Furthermore, Rhodamine uptake and Hoechst dye labeling has been used to select stem cell populations from BM, CB, mesenchymal, muscle, and adult brain (Kim et al., 2002; Bhattacharya et al., 2003; Migishima et al., 2003; Parmar et al., 2003). The side population (SP) which is demonstrated by low uptake of Hoechst dye 33342 represents the highest capability of self-renewing and pluripotency. Hoechst dye uptake is regulated by a membrane transporter ABCG2 and the SP population is defined as the expression of ABCG2 protein (Zhou et al., 2001; Scharenberg et al., 2002). ABCG2 protein is also expressed specifically in neural stem cells and decreases in expression when precursor cells differentiate (Cai et al., 2002).
Evidence of Ectodermal Cell Transdifferentiation from Human Hematopoietic Cell Lineage.
There are increasing reports of BM stroma derived progenitors differentiating into neural cells since these cells were first reported to show differentiation into muscle, glia, and hepatocytes in mouse (Azizi et al., 1998; Ferrari et al., 1998; Petersen et al., 1999). In vitro evidence of neuron specific proteins inductions, such as nestin, neuron-specific nuclear protein (NeuN), and glial acidic fibrillary protein (GFAP) in cells derived from human and rodent BM stromal cells were reported after stimulation with retinoic acid, epidermal growth factor (EGF), or brain derived neurotrophic factor (BDNF) (Sanchez-Ramos et al., 2000). Among non-mesenchymal hematopoietic progenitors, several reports have shown that human CB mononuclear cells including separated CD133+ cells were induced to express neuronal and glial markers in vitro such as beta-tublin III, GFAP after exposure to basic fibroblast growth factor (bFGF) and hEGF, and also Musashi-1 after retinoic acid and nerve growth factor (NGF) exposure (Sanchez-Ramos et al., 2001; Bicknese et al., 2002).
Evidence of Endodermal Cell Transdifferentiation from Hematopoietic Cell Lineage.
Previously, hepatocytes were thought to be transformed from infused BM cells in a mouse model (Lagasse et al., 2000), but it was found to be caused by cell fusion in that particular liver regeneration model (Wang et al, 2003b). Others also found that myelomonocytic cells from BM HSC source were the major source of hepatocyte fusion partners (Camargo et al, 2004). CB cells isolated from a lineage positive cell depletion procedure followed by a week of suspension culture formed an adherent cell population which were found to express markers for hepatic cells after further incubation with hepatocyte growth medium (McGuckin et al, 2005). In vivo evidence of hepatocyte-like cell development in the liver treated with CCl4 in immune deficient mice after CB CD34+.CD38-CD7− transplant was reported (Wang et al, 2003a) and more recently in a non-injury model using fetal sheep, human hepatocytes were generated through BM reconstitution of fetal sheep by human HSC, including CD34+/Lin−, CD34−/Lin−, CD34+/Lin−/CD38−, CD34−/Lin−/CD38−, CD34+/Lin−/CD133+, CD34+/Lin−/CD133− derived from either BM, peripheral blood, or CB (Almeida-Porada et al, 2004).
Harvesting and Banking of Other Tissue Types from the Placenta.
The placenta as a valuable source of a wide variety of tissues (other than cord blood) that can be harvested, banked, and transplanted has been gaining increasing acceptance within the medical community (Parolini et al., 2008). For example, a part of the placenta called the amnion, or the outer membrane of the amniotic sac, is comprised of cells that have strikingly similar characteristics to embryonic stem cells, including the ability to express two key genes that give embryonic stem cells their unique capability for developing into any kind of specialized cell. Amniotic epithelial cells could in fact be directed to form liver, pancreas, heart and nerve cells under the right laboratory conditions. Another example is Wharton's Jelly composed of primitive connective tissue of the umbilical cord. Wharton's jelly stem cells (WJSCs) have significant therapeutic potential because large number of cells are easily isolated and may be better tolerated following transplantation because of their low immunogenicity and immune suppression. The cells are a potential important tool for tissue engineering, cell and gene therapy for various genetic diseases and acquired diseases since WJSCs can be induced to form adipose tissue, bone, cartilage, skeletal muscle, cardio myocyte-like cells and neural cells. WJSCs could be used to treat protein deficiencies, disorders of bone and cartilage, cardiac diseases, bone marrow stromal disorders, neurological diseases such as Parkinson's disease, multiple sclerosis, cerebrovascular accidents (stroke) and even cerebral palsy. Finally, placental tissues have been used directly in the treatment of burned and ulcerated skin and conjunctival defects. It is noted that amniotic membranes have many beneficial properties including anti-inflammatory, bacteriostatic, analgesia, wound healing, etc.